Method of compensating and calibrating a flow cytometer, and microbead standards kit therefor

• US 4,857,451 A
• Filed: 12/04/1987
• Issued: 08/15/1989
• Est. Priority Date: 12/24/1984
• Status: Expired due to Term

• Current Assignee: Bangs Laboratories Incorporated (Polysciences Incorporated)
• Original Assignee: Flow Cytometry Standards Corporation
• Inventors: Schwartz, Abraham
• Primary Examiner(s): Richman, Barry S.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US07/128,786
• Time in Patent Office: 620 Days
• Field of Search: 436/10, 436/19, 436/800, 435/7, 526/273, 526/320, 526/328, 526/328.5, 526/181, 526/201, 526/88, 526/332, 424/3, 523/202, 523/205, 523/201, 523/223, 525/327.3, 525/330.3, 525/330.5, 525/343, 525/351, 525/379, 525/382, 525/384, 525/284, 525/350, 524/461, 524/464, 524/558, 524/853, 524/458, 428/407, 428/516, 428/402, 252/301.35, 252/408.1, 521/60, 521/149, 250/461.2, 356/39
• US Class Current: 435/7.24
• CPC Class Codes: G01N 15/1012 Calibrating particle analysers; References therefor
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1014 Constitution of reference particles
• CPC Class Codes: G01N 2015/1018 Constitution of reference particles
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]

Abstract

A kit of highly uniform microbead standards for flow cytometer alignment, compensation, and calibration, comprising a blank and/or auto-fluorescent microbead population, together with two or more series of calibrated microbead populations which match the fluorescence spectra of labeled samples to be measured on the flow cytometer. Also disclosed is a corresponding method to align, compensate, and calibrate a flow cytometer so as to make measurements on corresponding samples comparable and independent of the specific instrument and instrument settings.

Claims

1. A microbead standards kit for alignment, compensation, and calibration of a flow cytometer for subsequent measurement of a selected fluorescently labeled sample, said kit comprising:

(a) a population of microbeads characterized by a same fluoresence spectra and fluoresence intensity as a selected fluorescently unlabeled sample prior to the selected fluorescently unlabeled sample being fluorescently labeled to yield a selected fluorescently labeled sample, said microbead population being selected from the group consisting of fluorescently unlabeled microbeads and auto-fluorescent microbeads;

(b) populations of microbeads which are labeled with at least two fluorescent dyes such that said labeled microbead populations comprise microbeads which are characterized by fluorescence intensity levels registerable in multiple fluorescence channels of a flow cytometer, each same labeled population comprising a series of sub-populations of said labeled microbeads characterized by differing selected levels of fluorescence intensity to substantially encompass a range of fluorescence intensity of said selected fluorescently labeled sample to be measured by a flow cytometer, when said selected fluorescently labeled sample is fluorescently labeled with a same fluorescent dye;

(c) said microbead populations (a) and (b) being constituted by highly uniform same sized microbeads having a coefficient of variation of diameter of about 2% or less, with said size of said microbeads being substantially equivalent to a size of said selected fluorescently labeled sample to be measured by a flow cytometer; and

(d) container means enclosing said microbead populations (a) and (b).

2. A microbead standards kit according to claim 1, wherein said highly uniform size microbeads have a diameter in the range of from about 2 to about 20 microns.

3. A microbead standards kit according to claim 1, wherein said microbead population (b) comprises a first sub-population comprising microbeads labeled with a first fluorescent dye, and a second sub-population comprising microbeads labeled with a second fluorescent dye.

4. A microbead standards kit according to claim 1, wherein said microbead population (b) comprises microbeads which are multiply labeled with different fluorescent dyes.

5. A microbead standards kit according to claim 3, wherein said first fluorescent dye is fluorescein, and said second fluorescent dye is phycoerythrin.

6. A microbead standards kit according to claim 4, wherein said different fluorescent dyes comprise fluorescein and phycoerythrin.

7. A microbead standards kit according to claim 3, wherein said first fluorescent dye is Texas Red, and said second fluorescent dye is allophycocyanine.

8. A microbead standards kit according to claim 4, wherein said different fluorescent dyes comprise Texas Red and allophycocyanine.

9. A microbead standards kit according to claim 1, comprising a microbead population (a) of fluorescently unlabeled microbeads.

10. A microbead standards kit according to claim 1, wherein said microbead population (a) comprises auto-fluorescent microbeads.

11. A microbead standards kit according to claim 10, wherein said auto-fluorescent microbeads are labeled with riboflavin.

12. A method of aligning, compensating, and calibrating a flow cytometer comprising multiple fluorescence channels including F11 and F12 fluorescence channels, and forward and right angle scatter channels, a fluorescence excitation source, photomultiplyer tubes and emission barrier filters for said multiple fluorescence channels, and amplifier and gain setting means, for subsequent measurement of a selected fluorescently labeled sample, said method comprising the steps of:

(i) providing a microbead standards kit comprising;

(a) a population of microbeads characterized by a same fluorescence spectra and fluorescence intensity as a selected fluorescently unlabeled sample prior to the selected fluorescently unlabeled sample being fluorescently labeled to yield a selected fluorescently labeled sample, said microbead population being selected from the group consisting of fluorescently unlabeled microbeads and auto-fluorescent microbeads;

(b) populations of microbeads which are labeled with at least two fluorescent dyes such that said labeled microbead populations comprise microbeads which are characterized by fluorescence intensity levels registerable in multiple fluorescence channels of a flow cytometer, each same population comprising a series of sub-populations of said labeled microbeads characterized by differing selected levels of fluorescence intensity to substantially encompass a range of fluorescence intensity of said selected fluorescently labeled sample to be measured by a flow cytometer, when said selected fluorescently labeled sample is fluorescently labeled with a same fluorescent dye;

(c) said microbead populations (a) and (b) being constituted by highly uniform same size microbeads having a coefficient of variation of diameter of about 2% or less, with said size of said microbeads being substantially equivalent to the size of said selected fluorescently labeled sample to be measured by a flow cytometer; and

(d) container means enclosing said microbead populations (a) and (b);

(ii) running a first population of fluorescent microbeads of said microbead population (b) detectable in said F11 fluorescence channel of said flow cytometer, and aligning and focusing said flow cytometer such that a dot plot or histogram resulting from said first population of microbeads will have a maximum intensity and a minimum distribution in said forward and right angle scatter and F11 fluorescence channels;

(iii) repeating step (ii) with a second microbead population from said microbead population (b) labeled with a different fluorescent dye detectable in said F12 fluorescence channel of said flow cytometer;

(iv) running said microbead population (a) on said flow cytometer, and said adjusting fluorescence channel photomultiplyer tube voltages and gains of said flow cytometer to position a resulting dot plot or histogram near an origin of an axis of each said fluorescence channel, and setting boundary gates in each said fluorescence channel to indicate fluorescence intensity of said microbead population (a);

(v) mixing said first and second fluorescent microbead population used in steps (ii) and (iii) with the microbead population (a) to form a fluorescent microbead adjustment mixture;

(vi) running the fluorescent microbead adjustment mixture on said flow cytometer and adjusting compensation circuits of said flow cytometer so that a fluorescence signal from each of the said first and second microbead populations registers as positive fluorescence in a respective primary fluorescent channel and as non-fluorescent, matching an intensity level of said microbead population (a) in all other secondary fluorescence channels;

(vii) running on said flow cytometer microbead populations (b) labeled with fluorescent dyes, without changing any flow cytometer settings, to determine a modal peak channel of a series of microbead populations labeled with fluorescent dyes at varying specific levels of fluorescent intensity;

(viii) constructing a calibration plot of equivalent soluble fluorescent dye molecules per microbead as a function of fluorescence intensity channel of said flow cytometer;

(ix) running said fluorescently unlabeled microbead population (a) on said flow cytometer and determining a position of a modal peak thereof for each said fluorescence channel to determine a sensitivity of each said fluorescence channel of said flow cytometer; and

(x) determining on said calibration plot a position of a modal peak of the auto-fluorescent microbead population (a) for each said fluorescent channel to indicate a threshold above which fluorescence intensity of said selected fluorescently labeled sample may be measured by the said fluorescence channels of said flow cytometer.

13. A method according to claim 12, comprising repeating step (ii) for each of said fluorescent channels of said flow cytometer.

14. A method according to claim 12, further comprising running on said flow cytometer microbead populations (b) labeled with at least two fluorescent dyes to confirm capability of said flow cytometer for simultaneous fluorescence detection for samples labeled with a corresponding combination of said fluorescence dyes.

15. A method according to claim 12, wherein said fluorescent dye comprises at least one member of the group consisting of fluorescein, phycoerythrin, Texas Red, and allophyacocyanine.

16. A method according to claim 12, wherein said microbead population (a) comprises microbeads labeled with riboflavin at sufficient concentration to match fluorescence spectra and fluorescence intensity of said sample.

17. A method according to claim 12, comprising constructing said calibration plot as a log-log plot, wherein said amplfier means of said flow cytometer is a linear amplifier.

18. A method according to claim 12, wherein said calibration plot is constructed as a semi-log plot, and said amplifier means of said flow cytometer is a log amplifier.

19. A method of analyzing a selected sample on a flow cytometer, comprising measuring said sample on said flow cytometer after said flow cytometer has been aligned, compensated, and calibrated in accordance with the method of claim 12.

20. A method according to claim 19, wherein said sample comprises T-cells.

21. A method according to claim 20, wherein said T-cells are stained with fluorescent dyes comprising fluorescein isothiocyanate and phycoerythrin.

22. A method according to claim 21, wherein said T-cells are stained with said fluorescent dyes conjugated with monoclonal antibodies to said T-cells.